Proliferation and differentiation of cells within multicellular organisms is controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include the steroid hormones (e.g., estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGP), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO), insulin and calcitonin.
Hormones and growth factors influence cellular metabolism by binding to receptors. Receptors may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of receptors are soluble molecules, such as certain transcription factors.
Insulin belongs to a group of protein/polypeptide hormones. Insulin increases the rate of synthesis of glycogen, fatty acids, and proteins and stimulates glycolysis. It also promotes the transport of glucose, some other sugars, and amino acids into muscle and fat cells. The mature form of insulin consists of a 30 amino acid residue B chain, that is at the N-terminus of the propeptide form, and a 21 amino acid residue A chain, that is C-terminal. Proinsulin also contains a connecting peptide, C-peptide, between the B chain and A chain that is cleaved out to form mature insulin. The B chain and A chain are covalently joined by two disulfide bonds. The B-chain, C-peptide, A-chain motif is found in several other proteins including, relaxin (U.S. Pat. No. 4,835,251, incorporated herein by reference), insulin-like growth factors (IGF) I and II (Bang and Hall, In xe2x80x9cInsulin-like Growth Factorsxe2x80x9d, P. N. Schofield (eds.), 151-177, Oxford Univ. Press, Oxford; incorporated herein by reference), Leydig factor (Bullesbach et al., J. Biol. Chem. 270:16011-16015, 1995; incorporated herein by reference), and early placenta insulin-like factor (EPIL; Chassin et al., Genomics 29:465-470, 1995, incorporated herein by reference). Unlike the other members of the insulin superfamily, IGF I and IGF II have D and E domains that are cleaved post-translationally. Cysteines that are involved in disulfide bonds are conserved in all the members of the family and play a role in the tertiary structure of the molecules.
Spermatogenesis is the process by which a germ cell proceeds through multiple stages of differentiation, and culminates in the formation of a terminally differentiated cell with a unique function. Hematopoiesis can be used as a paradigm for understanding spermatogenesis, and while there are striking parallels between what is known about hematopoiesis and spermatogenesis, the maturation of spermatogonia (germ cells) is less clearly understood than the maturation of hematopoietic stem cells. Particularly deficient is an understanding of factors that regulate the maturation process in spermatogenesis. Recent evidence suggests that some cytokines involved in the progression of stem cells of the hematopoietic lineage to fully differentiated cells are also involved in sperm cell maturation. In a fashion similar to cytokine action in hematopoiesis, these cytokines are thought to act at specific stages in the germ cell""s maturation. For example, stem cell factor (also known as Steel factor and c-kit ligand) mRNA is expressed in spermatogonia (Manova et al., Development 110:1057-1066, 1990), and administration of a monoclonal antibody to stem cell factor to adult or prepubertal mice causes depletion of differentiating spermatogonia but has no effect on the non-differentiating spermatogonia, or spermatocytes (Yoshinaga et al., Development 113:689-699, 1991). Other cytokines that have been associated with spermatogenesis include IL-1, IL-6 and xcex2-TGF (Sharp, Regulation of Spermatogenesis, in Knobil and Neil (ed.), Physiol. Reproduction, (2nd ed.), Raven, N.Y., 1994).
Because growth factors have had an enormous impact on our understanding of and ability to treat metabolic and cellular disorders, discovery of new factors is important. A new growth factor found in testis is particularly important for elucidating the spermatogenic process and potentially affecting the outcome of that process.
The present invention provides an isolated testis-specific insulin homolog polypeptide selected from the group consisting of: (a) polyeptides comprising a sequence of amino acids encoded by the nucleotide sequence as shown in SEQ ID NO: 1; (b) allelic variants of (a); and (c) testis-specific insulin homolog polypeptides which are at least 85% identical to (a) or (b).
In another aspect, the present provides an isolated polynucleotide molecule selected from the group consisting of: (a) DNA molecules encoding a testis-specific insulin homolog polypeptide and comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 1 to nucleotide 564; (b) allelic variants of (a); (c) DNA molecules encoding a testis-specific insulin homolog polypeptide which are least 85% identical in nucleotide sequence to (a) or (b); and (e) degenerate nucleotide sequences encoding a testis-specific insulin homolog polypeptide.
In another aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcriptional promoter; a DNA segment selected from the group consisting of: (a) DNA molecules encoding a testis-specific insulin homolog polypeptide and comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 1 to nucleotide 564; (b) allelic variants of (a); (c) DNA molecules encoding a testis-specific insulin homolog polypeptide and which are least 85% identical in nucleotide sequence to (a) or (b); and a transcriptional terminator.
In another aspect, the present invention provides a cultured cell into which has been introduced an expression vector comprising the following operably linked elements: a transcriptional promoter; a DNA segment selected from the group consisting of: (a) DNA molecules encoding a testis-specific insulin homolog polypeptide and comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 1 to nucleotide 564; (b) allelic variants of (a); (c) DNA molecules encoding a testis-specific insulin homolog polypeptide which are least 85% identical in nucleotide sequence to (a) or (b); and a transcriptional terminator, and wherein the cell expresses a testis-specific insulin homolog polypeptide encoded by the DNA segment.
In another aspect, the present invention provides a method for producing a testis-specific insulin homolog polypeptide comprising culturing a cell into which has been introduced a first expression vector comprising the following operably linked elements: a transcriptional promoter; a DNA segment selected from the group consisting of: (a) DNA molecules encoding a testis-specific insulin homolog polypeptide and comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 1 to nucleotide 564; (b) allelic variants of (a); (c) DNA molecules encoding a testis-specific insulin homolog polypeptide which are least 85% identical in nucleotide sequence to (a) or (b); and a transcriptional terminator, whereby the cell expresses a testis-specific insulin homolog polypeptide encoded by the DNA segment, and recovering the testis-specific insulin homolog. Within one embodiment is provided a method for producing a Zins2 testis-specific insulin homolog polypeptide wherein the cell further comprises a second expression vector comprising the following operably linked elements: a transcriptional promoter; a DNA sequence encoding a prohormone convertase; and a transcriptional terminator. Within a related embodiment the prohormone convertase is selected from the group consisting of prohormone convertase 2, prohormone convertase 3, prohormone convertase 4 and furin.
Within another aspect, the invention provides an isolated, mature rat Zins2 testis-specific insulin homolog protein having: a B chain comprising amino acid residue 22 to amino acid residue 52 of SEQ ID NO:2; an A chain comprising amino acid residue 162 to amino acid residue 188 of SEQ ID NO:2; wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds. Within a related embodiment is provided an isolated, mature rat Zins2 testis-specific insulin homolog protein having: a B chain having the amino acid sequence from amino acid residue 23 to amino acid residue 51 of SEQ ID NO:2; an A chain having the amino acid sequence from amino acid residue 163 to amino acid residue 188 of SEQ ID NO:2, wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds.
The invention also provides an isolated, mature human Zins2 testis-specific insulin homolog protein having: a B chain comprising amino acid residue 20 to amino acid residue 54 of SEQ ID NO:13; an A chain comprising amino acid residue 172 to amino acid residue 213 of SEQ ID NO:13; wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds. Within a related embodiment is provided an isolated, mature human Zins2 testis-specific insulin homolog protein having: a B chain having the amino acid sequence from amino acid residue 21 to amino acid residue 53 of SEQ ID NO:13; an A chain having the amino acid sequence from amino acid residue 173 to amino acid residue 198 of SEQ ID NO:13, wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds.
Within another aspect, the invention provides a post-translationally modified Zins2 testis-specific insulin homolog polypeptide or polypeptide fragment having the amino acid sequence from amino acid residue 53 to amino acid residue 162 of SEQ ID NO:2; the amino acid sequence from amino acid residue 55 to amino acid residue 172 of SEQ ID NO:13 or the amino acid sequence from amino acid residue 201 to amino acid residue 213 of SEQ ID NO:13.
The invention also provides a pharmaceutical composition comprising an isolated testis-specific insulin homolog polypeptide as described above in combination with a pharmaceutically acceptable vehicle.
Also provided by the invention is an antibody that specifically binds to an epitope of an isolated testis-specific insulin homolog polypeptide as described above.
The invention further provides a binding protein that specifically binds to an epitope of an isolated testis-specific insulin homolog polypeptide as described above.
The invention additionally provides a binding protein that specifically binds to an epitope of a polypeptide selected from the group consisting of: (a) polypeptides comprising a sequence of amino acids encoded by the nucleotide sequence as shown in SEQ ID NO:12; (b) allelic variants of (a); and (c) testis-specific insulin homolog polypeptides which are at least 85% identical to (a) or (b).
Within another aspect, the invention provides a method of enhancing viability of cryopreserved sperm for use in fertilization of an egg, wherein a Zins2 testis-specific insulin homolog polypeptide selected from the group consisting of: (a) polypeptides comprising a sequence of amino acids encoded by the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:12; (b) allelic variants of (a); and (c) Zins2 testis-specific insulin homolog polypeptides which are at least 85% identical to (a) or (b); is added to sperm, an egg, an egg-sperm mixture, prior to fertilization.
Within another aspect, the invention provides a method of enhancing sperm motility wherein a Zins2 testis-specific insulin homolog polypeptide selected from the group consisting of: (a) polypeptides comprising a sequence of amino acids encoded by the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:12; (b) allelic variants of (a); and (c) Zins2 testis-specific insulin homolog polypeptides which are at least 85% identical to (a) or (b); is added to sperm, an egg, an egg-sperm mixture, prior to fertilization. Within a related embodiment is provided a method of enhancing sperm motility wherein the Zins2 testis-specific insulin homolog is added to sperm following cryopreservation.
Within another aspect, the invention provides a method of enhancing an egg-sperm interaction wherein a Zins2 testis-specific insulin homolog polypeptide selected from the group consisting of: (a) polypeptides comprising a sequence of amino acids encoded by the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:12; (b) allelic variants of (a); and (c) Zins2 testis-specific insulin homolog polypeptides which are at least 85% identical to (a) or (b); is added to sperm, an egg, an egg-sperm mixture prior to fertilization.
Within anther aspect, the invention provides a method of enhancing fertilization during assisted reproduction wherein a Zins2 testis-specific insulin homolog polypeptide selected from the group consisting of: (a) polypeptides comprising a sequence of amino acids encoded by the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:12; (b) allelic variants of (a); and (c) Zins2 testis-specific insulin homolog polypeptides which are at least 85% identical to (a) or (b); is combined with sperm, an egg, an egg-sperm mixture prior to fertilization of the egg. Within one embodiment is provided a method of enhancing fertilization wherein the assisted reproduction is artificial insemination. Within a related embodiment is provided a method of enhancing fertilization wherein the assisted reproduction is in vitro fertilization.
Also provided by the invention is a method of contraception wherein an antagonist of a Zins2 testis-specific insulin homolog polypeptide selected from the group consisting of: (a) polypeptides comprising a sequence of amino acids encoded by the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:12; (b) allelic variants of (a); and (c) Zins2 testis-specific insulin homolog polypeptides which are at least 85% identical to (a) or (b); is administered to a recipient to prevent fertilization of an egg. Within a related embodiment is provided a method of contraception according wherein the antagonist is an anti-Zins2 binding protein.
The invention further provides a method of immunocontraception wherein a vaccine containing a Zins2 testis-specific insulin homolog polypeptide selected from the group consisting of: (a) polypeptides comprising a sequence of amino acids encoded by the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:12; (b) allelic variants of (a); and (c) Zins2 testis-specific insulin homolog polypeptides which are at least 85% identical to (a) or (b); is administered to a recipient to prevent fertilization of an egg.
The invention also provides an isolated, mature rat Zins2 testis-specific insulin homolog protein according to claim 8 having: a B chain having the amino acid sequence from amino acid residue 23 to amino acid residue 51 of SEQ ID NO:2; an A chain having the amino acid sequence from amino acid residue 163 to amino acid residue 188 of SEQ ID NO:2, wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds.
Also provided is an isolated, mature human Zins2 testis-specific insulin homolog protein having: a B chain comprising amino acid residue 20 to amino acid residue 54 of SEQ ID NO:13; an A chain comprising amino acid residue 172 to amino acid residue 213 of SEQ ID NO:13; wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds.
Within another aspect the invention provides an isolated, mature rat zins2 testis-specific insulin homolog protein consisting of: a B chain having the amino acid sequence from amino acid residue 24 to amino acid residue 51 of SEQ ID NO:2; an A chain having the amino acid sequence from amino acid residue 163 to amino acid residue 188 of SEQ ID NO:2, wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds. Within a related embodiment the mature rat Zins2 testis-specific insulin homolog protein consists of: a B chain having the amino acid sequence from amino acid residue 24 to amino acid residue 52 of SEQ ID NO:2; an A chain having the amino acid sequence from amino acid residue 163 to amino acid residue 188 of SEQ ID NO:2, wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds.
The invention also provides an isolated, mature human Zins2 testis-specific insulin homolog protein consisting of: a B chain comprising amino acid residue 23 to amino acid residue 53 of SEQ ID NO:13; an A chain comprising amino acid residue 173 to amino acid residue 198 of SEQ ID NO:13; wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds. Within a related embodiment the mature human Zins2 testis-specific insulin homolog protein consists of: a B chain comprising amino acid residue 23 to amino acid residue 54 of SEQ ID NO:13; an A chain comprising amino acid residue 173 to amino acid residue 198 of SEQ ID NO:13; wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds. Within another embodiment the mature human Zins2 testis-specific insulin homolog protein consists of: a B chain comprising amino acid residue 23 to amino acid residue 54 of SEQ ID NO:13; an A chain comprising amino acid residue 173 to amino acid residue 199 of SEQ ID NO:13; wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds. Within yet another embodiment the mature human Zins2 testis-specific insulin homolog protein consists of: a B chain comprising amino acid residue 23 to amino acid residue 54 of SEQ ID NO:13; an A chain comprising amino acid residue 173 to amino acid residue 200 of SEQ ID NO:13; wherein the B chain and A chain are joined by inter- and intra-chain disulfide bonds.
Within another aspect the invention provides a method for detecting Zins2 post-translationally modified polypeptides or polypeptide fragments comprising: exposing a solution possibly containing Zins2 post-translationally modified polypeptides or polypeptide fragments to an antibody attached to a solid support, wherein said antibody binds to a first epitope of a Zins2 post-translationally modified polypeptide or polypeptide fragment; washing said immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a Zins2 post-translationally modified polypeptide or polypeptide fragment, wherein the second antibody is associated with a detectable label; and detecting the detectable label. Levels of Zins2 post-translationally modified polypeptides or polypeptide fragments in serum or biopsy are then correlated to tumor activity. Within a one embodiment the Zins2 post-translationally modified polypeptides or polypeptide fragments are selected from the group consisting of: a) a polypeptide consisting of the amino acid sequence from amino acid residue 53 to amino acid residue 162 of SEQ ID NO:2; b) a polypeptide consisting of the amino acid sequence from amino acid residue 55 to amino acid residue 172 of SEQ ID NO:13; c) a polypeptide consisting of the amino acid sequence from amino acid residue 201 to amino acid residue 213 of SEQ ID NO:13; and d) polypeptide fragments of a, b, or c.
Also provided is a method for detecting nucleic acid sequences encoding Zins2 post-translationally modified polypeptides or polypeptide fragments comprising: incubating a single-stranded probe molecule with RNA isolated from a biological sample under conditions of temperature and ionic strength that promote base pairing between the probe and target Zins2 RNA species; separating unbound probe from hybridized molecules; and detecting the presence of probe-Zins2 RNA hybrids. Within one embodiment the single-stranded probe molecule is at least 5 consecutive nucleotides selected from the group consisting of: a) nucleotides 157-487 of SEQ ID NO:1; b) nucleotides 179-532 of SEQ ID NO:12; and c) nucleotides 617-655 of SEQ ID NO:12.